1. Field of the Invention
This invention generally relates to human lipid metabolism, particularly to apolipoproteins, genes encoding these apolipoproteins, related proteins, and their mutations and polymorphisms as they relate to cardiovascular, coronary and other diseases.
2. Description of the Related Art
Cardiovascular diseases are the number one cause of death in Western societies. Studies repeatedly show that individuals with high levels of very low-density lipoprotein, (VLDL) and/or low levels of high density lipoprotein (HDL) have significantly increased chances of developing cardiovascular disease. It has been established that strategies to reverse the levels of these lipoprotein particles will lower disease risk in susceptible individuals.
Lipoproteins function as transport vehicles for triacylglycerols (triglycerides), cholesterol and other lipids. These complexes solubilize highly hydrophobic lipids, and regulate entry and exit of particular lipids at specific targets. Lipoproteins form micelle-like particles that consist of a nonpolar core of triacylglycerols, more commonly known as triglycerides, and cholesteryl esters surrounded by a coating of protein, phospholipids, and cholesterol. The lipoproteins are classified according to density. Lipoprotein particles are composed of lipids and proteins and are such particles as chylomicrons, very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). (Voet and Voet, Biochemistry, 1990; Stryer, Biochemistry, 1995). The protein components of lipoproteins are known as apolipoproteins.
Van der Vliet, H N et al., report on a gene that shares homology to APOAV and note its increase in expression following rat liver hepatectomy in J Biol Chem. Nov. 30, 2001;276(48):44512-20. The rat (GenBank Accession Nos. AF202888 and AF202887), mouse (GenBank Accessions No. AF327059) and human (GenBank Accessions Nos. AF202890 and AF202889) versions of these sequences were deposited in GenBank and entitled, Regeneration-associated protein 3 (Rap 3) mRNA, complete cds. Rap3 is noted in GenBank as an “apolipoprotein-like serum protein; concentration elevated after a 70% partial hepatectomy” in rats.
The human genomic region containing the DNA sequence for APOAV was sequenced by the Human Genome Project and deposited in GenBanK under Accesssion Numbers AC007707 and/or AC074203. These deposits cover approximately 200 kb of human genomic DNA. The deposits are associated with clustered 11q23 and 22q11 breakpoints, but no coding regions are described. Computational analyses indicate the previously described APOA-I, APOC-III, and APOA-IV are contained within this interval.
The GenBank Accession Number AC007707 sequence shows the opposite strand (reverse complement) of the sequences of the present invention. The reverse complement of AC007707 was used as the starting point of the present invention for finding the APOAV gene and coding sequence.
Yen et al., in PCT Publication WO 01/007803, entitled “Apolipoprotein A-IV-related protein: Polypeptide, polynucleotide sequences and bi-allelic markers thereof” describe a gene corresponding to the present APOAV. The gene is described as encoding an apolipoprotein A-IV-related protein (AA4RP) as well as regulatory sequences at the 5′ and 3′ end. Also disclosed are biallelic markers of the AA4RP gene useful in genetic analysis. However, Yen et al. describe their biallelic markers differently than the SNP's of the present invention. They disclose no description of any known linkages between these markers and any known disease phenotype.
Several human cDNA sequences derived from the APOAV gene have been previously disclosed in GenBank. A sequence file generated by the NCBI annotation project in July 2001 (transcript version 1) is disclosed as human mRNA/cDNA sequence, XM—052110. A second sequence file generated by the NCBI annotation project identifies in July 2001 (transcript version 2) can be found as XM—052109. AF202890 (called RAP3) is a third cDNA sequence that is related to the van der Vliet et al. Human ortholog of rat liver regeneration associated protein (transcript version 1). AF202889 (called RAP3) is the fourth cDNA sequence related to the van der Vliet et al. No. AF401201and was made public on 7 Oct. 2001.
Other related sequences include mouse mRNA/cDNA sequence (AF327059), called RAP3, which is the sequence identified as the mouse ortholog of rat liver regeneration associated protein. Three publicly generated mouse full-length cDNAs for apoAV are found under the following Accession Nos.: AK004903 (transcript version 2), BC011198 (transcript version 2), and AK004936 human ortholog of rat liver regeneration associated protein (transcript version 2).
Human protein sequences were predicted from mRNA sequences AF202890 and AF202889 and called AAF25662 and AAF25661, which correspond to the APOAV protein, which can be found under NP—443200 in NCBI.
The mouse genomic region (which includes additional genes sequenced and used used to create the knock-out mice described herein) is SEQ ID NO: 7 in this application. This sequence was deposited in GenBank under GenBank Accession (transcript version 1). The mouse RAP3 protein (GenBank Accession No. AAG49600) is the protein sequence predicted from mRNA sequence AF327059.